The present invention relates to a method for determining an unknown optical path length and for validating the measurement obtained, in particular a traceable method for determining the path length of a volume such as a drop.
Metrology is a science which is concerned with measurement; specifically, it includes experimental and theoretical determinations in any field of technology. The international vocabulary of metrology is maintained by the International Organisation for Standardisation (ISO) and is currently in its third revision.
One of the core bases of metrology is metrological traceability, which is defined as the property of the result of a measurement or the value of a standard whereby it can be related to references, usually national or international standards, through an unbroken chain of comparisons all having stated uncertainties. In many countries, national standards for weights and measures are maintained by a National Metrology Institute (NMI) which provides the highest level of standards for the calibration or measurement traceability infrastructure in that country. For example, In the UK, the NMI is the National Physical Laboratory (NPL), in the US the NMI is called National Institute of Standards and Technology (NIST), in Germany the NMI is the Physikalisch-Technische Bundesanstalt (PTB) and in Canada the NMI is the NRC Institute for National Measurement Standards (NRC).
Typically, traceability is achieved by calibration which establishes the relation between the result shown in a measuring instrument and the value of a measured standard. Thus, calibration to a traceable standard can be used to determine whether an instrument is precise and accurate and it can also be used to determine whether the instrument has a bias.
Traceability can also be obtained from a derived unit back to one of the fundamental units of the International System of Units (SI) convention. In the present invention, a derived unit, specifically absorbance, is traced back to the metre.
The Beer-Lambert law states that there is a logarithmic dependence between the transmission or transmissivity (T) of light through a substance and the product of the absorption coefficient (α) of the same substance and the distance the light travels through the material, commonly referred to as the path length (l). Accordingly, the absorption coefficient (α) can be expressed as a product of either: (a) the extinction coefficient or molar absorptivity of the substance (ε) and the concentration (c) of absorbing species in the material or (b) an absorption cross-section (σ) and the density (N′) of absorbers. For liquids, these relations are usually written as:
                    T        =                              I                          I              0                                =                                    10                                                -                  α                                ⁢                                                                  ⁢                l                                      =                          10                                                -                  ɛ                                ⁢                                                                  ⁢                lc                                                                        (        1        )            
Wherein I0 is the intensity or power of the incident light and I is the intensity of the transmitted light.
Transmission (T) is expressed in terms of absorbance. For liquids, absorbance is defined as:
                    A        =                  -                                    log              10                        ⁡                          (                              I                                  I                  0                                            )                                                          (        2        )            
From the above, it can be deduced that absorbance becomes linear with the concentration (or number density of absorbers) as shown by the following equation:A=εlc=αl  (3)
According to the above equation, for a liquid, if the path length and the molar absorptivity or the absorption cross section are known, the concentration of a substance or the number density of absorbers can be calculated by measuring absorbance.
Typically, in analytical spectroscopy, a cuvette with a 1 cm path length is used in a standard system to simplify the above calculation. This allows the concentration of an unknown calculation to be deduced by measuring its absorbance and comparing it to a series of absorbance values of standard solutions shown in a standard curve.
However, in vertical spectrophotometry the path length varies from sample to sample. Further, the same problem may arise for very small volumes of sample in a horizontal system.
The present invention therefore seeks to allow an unknown path length to be determined by suing a high accuracy reference path length and certified reference solutions or material to achieve fully traceable measurements.
U.S. Pat. No. 5,959,738 (and its continuations—U.S. Pat. Nos. 6,188,476, 6,320,662, and 6,404,501) disclose a photometric method and a device for determining optical path lengths of liquid samples comprising analytes dissolved or suspended in a solvent by vertical spectrophotometry. This document also describes a method and apparatus for determining optical path length and sample concentration which provide accurate results. In this document, path length is calculated by measuring absorbance of the same sample at two different wavelengths and calculating the mathematical difference between the absorbance measure at the higher wavelength and the absorbance measurement at the lower wavelength and then dividing the obtained number by the mathematical difference between the absorbance measure at the higher wavelength and the absorbance measurement at the lower wavelength of a standard solution.
Although the methods and apparatus described in these documents are an improvement over other previous systems, the measurements are only comparative and are not suitable for working with the very small volumes required in biotechnology and pharmaceutical research.
WO2007/131945 discloses an apparatus having a source of electromagnetic radiation, a detector and a drop head comprising adapted to receive a drop of liquid and further adapted to be located, in use, in line with the source and the detector to allow analysis of the drop containing a sample. In this device the drop head is shaped to restrict the configuration of the drop so that surface tensions are more significant than gravity. This device is also an improvement over other prior art. However, it does not provide fully traceable results because each measurement cannot be validated to an accurate standard. Thus, the path length measurement obtained cannot be used as part of ISO-compliant protocols nor can it be used to obtain ISO-compliant results.
The present invention therefore seeks to provide a traceable method of determining an unknown optical path length.